1. Field of the Invention
The present invention relates to novel N-alkylacridan carboxyl derivatives which produce light. This invention further relates to an improved method of generating light chemically (chemiluminescence) by the action of a peroxidase enzyme and an oxidant such as hydrogen peroxide with a group of N-alkylacridan carboxyl derivatives. The invention also relates to a method of greatly increasing the amount of chemiluminescence produced from this process by the use of enhancers. The invention also relates to the use of this method to detect the peroxidase enzyme. The invention also relates to the use of this method to detect hydrogen peroxide. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting and nucleic acids by enzyme-linked nucleic acid probes. The method may also be used to detect DNA in DNA sequencing applications. The method may additionally be used to detect enzymes which generate hydrogen peroxide such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase and the like.
2. Description of Related Art
The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Further increases in assay sensitivity will expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. A way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of substrates which generate light with a higher efficiency or for a greater length of time.
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. To take better advantage of the beneficial properties of this enzyme in analysis, new chemiluminescent substrates which permit the detection of lower amounts of enzyme would be desirable. Specifically, substrates which generate higher levels of chemiluminescence via either a higher maximum intensity or a longer duration than compounds known in the art would be advantageous.
a. Oxidation of Acridan.
Oxidation of acridan by benzoyl peroxide in aqueous solution produced chemiluminescence with very low efficiency (O.sub.CL =3.times.10.sup.-7) and a mixture of products including acridine (S. Steenken, Photochem. Photobiol., 11, 279-283 (1970)). N-Methylacridan is oxidized electrochemically to N-methylacridinium ion (P. Hapiot, J. Moiroux, J. M. Saveant, J. Am. Chem. Soc., 112 (4), 1337-43 (1990); N. W. Koper, S. A. Jonker, J. W. Verhoeven, Recl. Trav. Chim. Pays-Bas, 104 (11), 296-302 (1985)). Chemical oxidation of N-alkylacridan compounds has been performed with ferricyanide ion (A. Sinha, T. C. Bruice, J. Am. Chem. Soc., 106(23), 7291-2 (1984)), certain quinones (A. K. Colter, P. Plank, J. P. Bergsma, R. Lahti, A. A. Quesnel, A. G. Parsons, Can. J. Chem., 62(9), 1780-4 (1984)), and lithium nitrite (O. N. Chupakhin, I. M. Sosonkin, A. I. Matern, G. N. Strogov, Dokl. Akad. Nauk SSSR, 250(4), 875-7 (1980)). Oxidation of an N-alkylacridan derivative has been performed photochemically with or without a flavin compound as co-oxidant (W. R. Knappe, J. Pharm. Sci., 67(3), 318-20 (1978); G. A. Digenis, S. Shakshir, M. A. Miyamoto, H. B. Kostenbauer, J. Pharm. Sci., 65(2), 247-51 (1976)).
Aryl and alkyl esters of 10-methylacridan-9-carboxylic acid undergo autoxidation to N-methylacridone in dipolar aprotic solvents under strongly basic conditions to produce chemiluminescence (F. McCapra, Accts. Chem. Res., 9(6), 201-8 (1976)). Chemiluminescence quantum yields ranged from 10.sup.-5 to 0.1 and were found to increase as the pK.sub.a of the phenol or alcohol leaving group decreased. Quantum yields in aqueous solution were significantly lower due to a competing non-luminescent decomposition of an intermediate. Addition of the cationic surfactant CTAB increased the apparent light yield 130-fold by preventing a competing dark reaction.
No reports exist on the use of peroxidase or other enzymes to oxidize acridans or substituted acridans. No reports exist on the generation of chemiluminescence from the reaction of acridans or substituted acridans with peroxidase or any other enzymes.
b. Chemiluminescent Oxidation of Acridinium Esters.
The chemiluminescent oxidation of aliphatic and aromatic esters of N-alkylacridinium carboxylic acid by H.sub.2 O.sub.2 in alkaline solution is a well-known reaction. The high chemiluminescence quantum yield approaching 0.1 has led to development of derivatives with pendant reactive groups for attachment to biological molecules. Numerous chemiluminescent immunoassays and oligonucleotide probe assays utilizing acridinium ester labels have been reported.
The use of acridinium esters (AE's), especially when labeled to a protein or oligonucleotide suffers from two disadvantages. The chief problem is limited hydrolytic stability. Acridinium ester conjugates decompose steadily at or slightly above room temperature. Depending on the substitution of the leaving group storage at -20.degree. C. may be required for extended storage.
A second disadvantage of acridinium esters is the tendency to add nucleophiles such as water at the 9-position to spontaneously form a pseudo-base intermediate which is non-luminescent and decomposes in a pH-dependent manner in a dark process. In practice the pH of solutions containing acridinium esters must be first lowered to reverse pseudo-base formation and then raised in the presence of H.sub.2 O.sub.2 to produce light.
Recently amides, thioesters and sulfonamides of N-alkylacridinium carboxylic acid have also been prepared and shown to emit light when oxidized under these conditions (T. Kinkel, H. Lubbers, E. Schmidt, P. Molz, H. J. Skripczyk, J. Biolumin. Chemilumin., 4, 136-139 (1989); G. Zomer, J. F. C. Stavenuiter, Anal. Chim. Acta, 227, 11-19 (1989)). These modifications of the leaving group only partially improve the storage stability performance.
A more fundamental limitation to the use of acridinium esters as chemiluminescent labels lies in the fact that when used as direct labels, only up to at most about 10 molecules can be attached to protein or oligonucleotide. Coupled with the quantum efficiency for producing a photon (.ltoreq.10%), an acridinium ester-labeled analyte can generate at most one photon of light. No further improvement in signal generating ability is possible.
An attempt to increase the number of acridinium ester molecules associated with an analyte in an immunoassay was made by constructing an antibody-liposome conjugate wherein the liposome contained an unspecified number of AE's (S. -J. Law, T. Miller, U. Piran, C. Klukas, S. Chang, J. Unger, J. Biolumin. Chemilumin., 4, 88-98 (1989)). Only a modest increase in signal was observed over a comparable assay using directly labeled AE's.
There is no known use of a peroxidase or other enzyme in conjunction with acridinium ester chemiluminescence.
c. Chemiluminescent Detection of Horseradish Peroxidase.
Amino-substituted cyclic acylhydrazides such as luminol and isoluminol react with H.sub.2 O.sub.2 and a peroxidase enzyme catalyst (such as horseradish peroxidase, HRP) under basic conditions with emission of light. This reaction has been used as the basis for analytical methods for the detection of H.sub.2 O.sub.2 and for the peroxidase enzyme. Various enhancers have also been employed in conjunction with the use of luminol to increase the intensity of light emitted. These include D-luciferin (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983)) and p-iodophenol and p-phenylphenol (G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead, Clin. Chem., 31, 1335 (1985)). To date, the only other chemiluminescent compound oxidized by a peroxidase enzyme and a peroxide is a hydroxy-substituted phthalhydrazide (Akhavan-Tafti co-pending U. S. patent application Ser. No. 965,231, filed Oct. 23, 1992).
The mechanism of oxidation of phthalhydrazides by the combination of a peroxide and a peroxidase enzyme is very complex and remains the subject of intense debate. This difficulty has hampered the development of new chemiluminescent reactions catalyzed by peroxidases. Nevertheless, the enzyme horseradish peroxidase has found use in enzyme immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as substrate (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983); G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead, Clin. Chem., 1335 (1985); G. H. Thorpe, S. B. Mosely, L. J. Kricka, R. A. Stott, T. P. Whitehead, Anal. Chim. Acta, 170, 107 (1985), and J. A. Matthews, A. Batki, C. Hynds, L. J. Kricka, Anal. Biochem., 151, 205, (1985)). Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are available.
Synthetic peptide-isoluminol derivatives such as t-Boc-alanylalanylphenylalanylisoluminolamide are substrates for the protease enzymes chymotrypsin, trypsin and thrombin. Reaction of compounds of this type with a protease enzyme liberates isoluminol which then can react with a peroxidase enzyme and H.sub.2 O.sub.2 to generate chemiluminescence. (B. R. Branchini, G. M. Salituro, in Bioluminescence and Chemiluminescence: Instrumental Applications, K. Van Dyke, ed., CRC Press, Boca Raton, Fla., Volume 2, pp. 25-39, (1985)).
Urdea U.S. Pat. No. 5,132,204 describes a stable 1,2-dioxetane which rapidly decomposes with emission of chemiluminescence after the consecutive removal of protecting groups by HRP and alkaline phosphatase from a phenol moiety. The doubly protected compound is, however, also chemiluminescent in the absence of enzyme through slow thermal decomposition or hydrolysis of the protecting group. No examples involving N-alkylacridan carboxyl derivatives were shown.